The broad long-term objective of this proposal is to establish heme oxygenase-1 (HO-1)-derived carbon monoxide (CO) as a biologically important gas that promotes homeostasis following arterial injury. We have recently demonstrated that gene transfer of HO-1 or the exogenous administration of CO blocks neointima formation following arterial injury, and that this is associated with a marked decrease in vascular smooth muscle cell growth and collagen deposition. We now propose to extend these studies and establish the significance and mechanism by which CO regulates endothelial cell (EC) function following arterial injury and in vascular disease. The central hypothesis of this proposal is that CO plays a critical role in promoting EC growth following arterial injury and that CO reverses endothelial dysfunction in hyperhomocysteinemia and diabetes. We further propose that CO mediates these effects via the activation of eNOS. In aim 1, we will examine the effect of endogenously derived or exogenously administered CO in regulating EC function and regrowth following arterial injury. These studies will investigate the effect of CO on EC proliferation, migration, apoptosis, and senescence, and determine whether the eNOS-mediated release of NO contributes to the biological actions of CO. We will also examine the mechanism by which CO regulates eNOS activity exploring possible transcriptional, postranscriptional, and posttranslational modes of regulation. In addition, we will investigate the effect of HO-1 gene transfer, HO-1 gene deletion, or CO administration on endothelial function and regrowth following carotid artery injury in rodents. In aim 2, we will determine whether the induction of HO-1 and CO synthesis in hyperhomocysteinemia functions in an adaptive manner to preserve endothelial function and blood pressure. In aim 3, we will investigate whether the dysregulation of CO synthesis in diabetes contributes to the development of endothelial dysfunction. In addition, we will examine whether restoration of endogenous CO synthesis or exogenous delivery of CO corrects endothelial function and regrowth following arterial injury. It is anticipated that these studies will establish the HO-1/CO system as a critical regulator of EC function, and will identify CO as a novel therapeutic modality in preventing endothelial dysfunction and vascular disease.